Establishment method of mature embryo callus induction and regeneration system of siberian wildrye seeds

Abstract

The invention discloses a method for establishing a mature embryo callus induction and regeneration system of siberian wildrye seeds, which comprises the following steps of: screening the siberian wildrye seeds collected in the field, removing fungi on the seeds, and disinfecting the siberian wildrye seeds; after the disinfected seeds are longitudinally cut, the seeds are sequentially put into an induction culture medium to be subjected to induction culture, a subculture medium to be subjected to subculture, a differentiation and proliferation culture medium to be subjected to differentiation and proliferation culture and a rooting culture medium to be subjected to rooting culture, and to-be-differentiated seedlings with the root length being 4-5 cm are obtained; moving the to-be-differentiated seedlings out of the rooting culture medium, closing the bottle for seedling hardening, cleaning roots, injecting tap water, opening the bottle for seedling hardening, and culturing by using sterilized vermiculite to obtain complete regenerated plants. According to the method, the growth of the plant callus is remarkably improved, rapid, efficient and genetically stable plant propagation is realized, and a basic experimental material is provided for molecular breeding of the Elsholtzia sibirica.

Description

Technical Field

[0001] The present invention belongs to the field of gene editing breeding, and particularly relates to a method for inducing callus from mature embryos of Elymus sibiricus seeds and establishing a regeneration system. Background Art

[0002] Elymus sibiricus L. belongs to the genus Elymus in the family Poaceae and is a perennial herb. It is rich in a large amount of protein and is the forage grass with the highest forage value in the genus Elymus. At the same time, Elymus sibiricus has strong regeneration ability and tillering ability, and has a high grass yield. The leaves of Elymus sibiricus are soft in texture and good in palatability, and are liked by all kinds of livestock. Elymus sibiricus is easy to establish, can grow well on barren, weakly acidic, slightly alkaline or humus-rich soils, and is extremely cold-resistant, and can overwinter safely at low temperatures of -30°C to -40°C. It is a forage grass with great cultivation value. Detection of fungi carried by Elymus sibiricus seeds collected from multiple places found that the fungus-carrying rate was 24% - 38%. Seed-borne fungi significantly reduced seed germination, inhibited seedling growth, and reduced the biomass of seedlings. Therefore, removing seed-borne fungi and improving the callus induction rate and growth rate are crucial.

[0003] With the emergence and maturity of various gene editing technologies, the process of plant breeding has been strongly promoted. However, only 11 plants in the Poaceae family have a genetic transformation system, which greatly affects the application of molecular biology breeding, making the breeding methods of most Poaceae plants still stay in the traditional breeding stage.

[0004] In summary, establishing an efficient and stable genetic transformation system for Elymus sibiricus can provide basic materials for enriching the germplasm resources of Elymus sibiricus and gene editing research. Summary of the Invention

[0005] To overcome the shortcomings and deficiencies of the prior art, the purpose of the present invention is to provide a method for inducing callus from mature embryos of Elymus sibiricus seeds and establishing a regeneration system.

[0006] The present invention is implemented as follows. A method for inducing callus from mature embryos of Elymus sibiricus seeds and establishing a regeneration system, the method comprising the following steps:

[0007] (1) Screening the Elymus sibiricus seeds collected in the wild to remove seed-borne fungi, and performing disinfection treatment;

[0008] (2) After longitudinally cutting the disinfected seeds, sequentially placing them in an induction medium for induction culture, a subculture medium for subculture, a differentiation and proliferation medium for differentiation and proliferation culture, and a rooting medium for rooting culture to obtain a to-be-differentiated seedling with a root length of 4 - 5 cm;

[0009] (3) Transfer the seedlings to be differentiated out of the rooting medium, acclimatize them in a closed bottle, then wash the roots and inject tap water, acclimatize them in an open bottle, and culture them with sterilized vermiculite to obtain complete regenerated plants.

[0010] Preferably, in step (1), the treatment for removing the seed-borne fungi is: placing the seeds in a drying oven at 65 °C for 14 d.

[0011] Preferably, in step (1), the disinfection treatment is: soaking the seeds successively in 50% H2SO4 for 10 min, 75% C2H5OH for 2 min, and 50% 84 for 20 min.

[0012] Preferably, in step (2), the process of induction culture is: longitudinally cut the seeds in half and place them in an induction medium with a pH of 5.8 - 5.9, and culture them for 31 - 35 d under dark conditions at a constant temperature of 26 ± 1 °C. Among them, the induction medium is: basic medium + 3.5 mg / L 2,4-D + 0.04 mg / L 6-BA.

[0013] Preferably, in step (2), the process of subculture is: placing the seeds in a subculture medium with a pH of 5.8 - 5.9, and culture them for 14 - 16 d under dark conditions at a constant temperature of 26 ± 1 °C; among them, the subculture medium is: basic medium + 3.5 mg / L 2,4-D + 0.04 mg / L 6-BA.

[0014] Preferably, in step (2), the process of differentiation and proliferation culture is: placing the seeds in a differentiation and proliferation medium with a pH of 5.7, and culture them under light conditions at a constant temperature of 26 ± 1 °C until the seedling length reaches 2 - 3 cm; among them, the differentiation and proliferation medium is: basic medium + 2 mg / L 6-BA + 0.5 mg / L NAA; the light conditions are: light culture, light intensity 4300 - 4800 Lux, and light duration 14 h·d -1 。

[0015] Preferably, in step (3), the process of rooting culture is: placing the seedlings in a rooting medium with a pH of 5.7, and culture them under light conditions at a constant temperature of 26 ± 1 °C until the root length reaches 4 - 5 cm; among them, the rooting medium is the basic medium, and the light conditions are: intensity 4300 - 4800 Lux and light duration 14 h·d -1 。

[0016] Preferably, the basic medium is: 4.33 g / L MS salts powder + 30 g / L maltose + 3.5 g / L phytagel.

[0017] Compared with the disadvantages and deficiencies of the prior art, the present invention has the following beneficial effects: when removing the pathogenic fungi on the seed coat of Elymus sibiricus, the growth of plant callus is significantly improved. Under strict aseptic conditions, the method of the present invention utilizes the totipotency of plant cells to achieve rapid, efficient and genetically stable plant propagation, providing basic experimental materials for the molecular breeding of Elymus sibiricus. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 The figure shows the callus on the 7th day after induction; among them, Figure A is the callus figure of the existing patent, and Figure B is the callus figure of the embodiment of the present invention;

[0019] Figure 2 The callus on the 40th day after induction; among them, Figure A is the callus figure of the existing patent, and Figure B is the callus figure of the embodiment of the present invention;

[0020] Figure 3 The differentiation of green seedlings from the callus of Elymus sibiricus in the embodiment of the present invention;

[0021] Figure 4 The rooting culture of Elymus sibiricus in the embodiment of the present invention;

[0022] Figure 5 The regenerated plants of Elymus sibiricus after transplantation in the embodiment of the present invention. DETAILED DESCRIPTION OF THE EMBODIMENTS

[0023] In order to make the objectives, technical solutions and advantages of the present invention clearer and more understandable, the present invention will be further described in detail below with reference to the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not used to limit the present invention.

[0024] The specific steps of the method of the present invention are as follows:

[0025] (1) Selection and dry heat sterilization of plant materials: Select seeds that are mold-free, disease-free, plump and of uniform size and put them into a kraft paper envelope bag. Seal the mouth of the envelope bag tightly with a clip and mark the species name and the date of putting into the oven on the envelope bag. Then put the envelope bag into an oven at 65 °C and set different treatment durations: 7d, 14d, 21d, 28d (the oven in the laboratory is opened for 12 hours every day).

[0026] (2) Treatment of plant materials: 10 mL of Elymus sibiricus seeds after dry heat treatment were placed in a 50 mL centrifuge tube. Set the seed disinfection combination method: 10 min of 50% H2SO4 + 2 min of 75% C2H5OH + 20 min of 50% 84. After disinfection with the first two chemical reagents, the seeds were washed 4 - 6 times with ultrapure water. After the last step of disinfection, the centrifuge tube containing the seeds was placed in a laminar flow hood, and the seeds were washed 7 - 9 times with sterile water in the laminar flow hood. The seeds were placed on sterile filter paper for the next operation. During the soaking process, the 50 mL centrifuge tube needs to be placed on a rotary shaker for mixing.

[0027] (3) Cut the disinfected Elymus sibiricus seeds in half and place them on the medium:

[0028] The basic medium: 4.33 g / L of MS salts powder + 30 g / L of maltose + 3.5 g / L of phytagel; autoclaved at 115 °C for 30 min; different concentration ratios of two plant hormones, 2,4-D and 6-BA, in the induction medium were tried, and 2.0 mg / L of 2,4-D and 0.05 mg / L of 6-BA, 2.5 mg / L of 2,4-D and 0.05 mg / L of 6-BA, 3.0 mg / L of 2,4-D and 0.05 mg / L of 6-BA, 3.5 mg / L of 2,4-D and 0.05 mg / L of 6-BA, 4.0 mg / L of 2,4-D and 0.05 mg / L of 6-BA, 4.5 mg / L of 2,4-D and 0.05 mg / L of 6-BA were set respectively. A total of seven ratios were found, and the callus induction rate was the highest with 3.5 mg / L of 2,4-D and 0.05 mg / L of 6-BA. Subsequently, the concentration of 6-BA was screened, and 3.5 mg / L of 2,4-D and 0.06 mg / L of 6-BA, 3.5 mg / L of 2,4-D and 0.05 mg / L of 6-BA, 3.5 mg / L of 2,4-D and 0.04 mg / L of 6-BA, 3.5 mg / L of 2,4-D and 0.03 mg / L of 6-BA were set respectively. The best concentration ratio of the two hormones was found to be 3.5 mg / L of 2,4-D and 0.04 mg / L of 6-BA. At this concentration ratio, the callus induction rate of Elymus sibiricus was the highest, reaching 96.5% (in a tissue culture method of Elymus plants disclosed in the existing patent (publication number CN 119256974 A), the highest callus induction rate was 94%).

[0029] Induction medium: basic medium + 3.5 mg / L of 2,4-D + 0.04 mg / L of 6-BA, PH = 5.7 - 5.8;

[0030] Subculture medium: basic medium + 3.5 mg / L of 2,4-D + 0.04 mg / L of 6-BA, PH = 5.7 - 5.8

[0031] Differentiation and proliferation medium: basal medium + 2 mg / L 6-BA + 0.5 mg / L NAA, PH = 5.7;

[0032] Rooting medium: basal culture medium.

[0033] (4) Treatment and induced differentiation of mature embryos: Place the Elymus sibiricus seeds disinfected in step (2) into the induction medium in step (3), and culture them in the dark in a tissue culture room at a constant temperature of 26 ± 1 °C for 31 - 35 d. During the dark culture, check in time whether there is fungal contamination of the seed belt. And count the contamination rate of miscellaneous bacteria and the callus induction rate. The morphology of the obtained callus is as Figure 1 B and Figure 2 shown in B. Transfer the non-polluted and well-growing callus into subculture for continuous culture. After 14 - 16 d of subculture, transfer it into the differentiation medium for light culture. The light intensity is 4300 - 4800 Lux, the light is on for 14 h every day, and the culture temperature is 26 ± 1 °C. Take pictures of the green seedlings differentiated from the Elymus sibiricus callus, as Figure 3 shown.

[0034] (5) When the length of the differentiated seedlings is 2 - 3 cm, transfer them into the rooting medium. Place them in a tissue culture room with a light intensity of 4300 - 4800 Lux, with light on for 14 h every day, and the culture temperature is 26 ± 1 °C. Take pictures of the differentiated seedlings in the rooting medium, as Figure 4 shown. When the roots of the differentiated seedlings grow to about 4 - 5 cm, move the seedlings out of the tissue culture room and acclimatize them in the dark with the bottle closed under natural light. After 2 d, wash the roots of the plants and then inject tap water, and acclimatize them with the bottle open under natural light. Then count the rooting rate. Open the culture bottle and plant them in vermiculite sterilized by a high-pressure steam sterilizer for culture to obtain complete plants, as Figure 5 shown.

[0035] Through experimental verification, the above experimental steps and the selection and ratio of experimental materials are all correct, and finally complete new plants can be obtained. Using the above-mentioned disinfection and sterilization methods. The contamination rate of Elymus sibiricus callus is between 5% and 2%, and the callus induction rate is between 77.3% and 96.5%. Its optimal combination is: put the seeds into a drying oven at 65 °C for 14 d and then take them out; adopt the seed disinfection method of 10 min of 50% H2SO4 + 2 min of 75% C2H6O + 20 min of 50% 84. The final fungal rate of the seed belt by the above method is 0%, the contamination rate is 3.4% - 5.2%, and the callus induction rate is 94.2% - 96.5%.

[0036] The specific process of the embodiment of the present invention is as follows.

[0037] Example 1. Influence of different durations of dry heat disinfection on the contamination rate

[0038] (1) Selection and preparation of plant materials: Select seeds that are mold-free, disease-free, plump, and of uniform size, and place them in a kraft paper envelope bag. Seal the opening of the envelope bag tightly with a clip, and mark the species name and the date of placing it in the oven on the envelope bag. Then place the envelope bag in an oven at 65 °C for different durations of treatment.

[0039] (2) Treatment of plant materials: Place 10 mL of dry-heat sterilized Elymus sibiricus seeds in a 50 mL centrifuge tube. Set the seed disinfection combination method: 10 min of 50% H2SO4 + 2 min of 75% C2H5OH + 20 min of 50% 84. After disinfection with the first two chemical reagents, wash the seeds 4 - 6 times with ultrapure water. After the last step of disinfection, place the centrifuge tube containing the seeds in a laminar flow hood and wash the seeds 7 - 9 times with sterile water in the laminar flow hood. Place the seeds on sterile filter paper for the next step. During the soaking process, place the 50 mL centrifuge tube on a rotary shaker.

[0040] (3) Cut the disinfected Elymus sibiricus seeds in half and place them on the culture medium, and count the contamination rate of the callus:

[0041] Oven placement duration (12 h per day) Pollution rate 7d 23％-25.7％ 14d 3.4％-5.2％ 21d 3.5％-4.3％ 28d 3.2％-4.8％

[0042] As the duration of placing in the oven increases, the viability of the seeds will correspondingly decrease. Finally, based on ensuring the seed viability, determine the oven placement duration to be 14 days.

[0043] Example 2. Effect of different 2,4-D concentrations on the callus induction rate

[0044] (1) Selection and preparation of plant materials: Select seeds that are mold-free, disease-free, plump, and of uniform size, and place them in a kraft paper envelope bag. Seal the opening of the envelope bag tightly with a clip, and mark the species name and the date of placing it in the oven on the envelope bag. Then place the envelope bag in an oven at 65 °C for different durations of treatment for 14 days.

[0045] (2) Treatment of plant materials: Place 10 mL of dry-heat sterilized Elymus sibiricus seeds in a 50 mL centrifuge tube. Set the seed disinfection combination method: 10 min of 50% H2SO4 + 2 min of 75% C2H5OH + 20 min of 50% 84. After disinfection with the first two chemical reagents, wash the seeds 4 - 6 times with ultrapure water. After the last step of disinfection, place the centrifuge tube containing the seeds in a laminar flow hood and wash the seeds 7 - 9 times with sterile water in the laminar flow hood. Place the seeds on sterile filter paper for the next step. During the soaking process, place the 50 mL centrifuge tube on a rotary shaker.

[0046] (3) Cut the disinfected Elymus sibiricus seeds in half and place them on the culture medium, and screen the concentration of the plant hormone 2,4-D in the culture medium:

[0047]

[0048]

[0049] Finally, the concentration of 2,4-D with the best callus induction rate is 3.5 mg / L.

[0050] Induction medium: basic medium + 3.5 mg / L 2,4-D + 0.05 mg / L 6-BA;

[0051] Subculture medium: basic medium + 3.5 mg / L 2,4-D + 0.05 mg / L 6-BA;

[0052] Differentiation and proliferation medium: basic medium + 2 mg / L 6-BA + 0.5 mg / L NAA;

[0053] Rooting medium: basic medium;

[0054] The said basic medium: 4.33 g / L MS salts powder + 30 g / L maltose + 3.5 g / L phytagel. Except for the pH value of the rooting medium being 5.7, the pH values of other media are 5.8 - 5.9.

[0055] (4) Treatment and induction of differentiation of mature embryos: Take the disinfected seeds in step (2), place them in the induction medium for 31 - 30 days, then transfer the non-contaminated and well-grown callus into the subculture medium for 14 days. Both the induction and subculture are carried out in the dark environment of the tissue culture room. The environmental temperature is 26 ± 1°C. Before transferring to the subculture medium, the contamination rate and callus induction rate are statistically recorded. The contamination rate is 3.4% - 5.2%, and the callus induction rate is 94.2% - 95.5%.

[0056] Example 3. Influence of different 6-BA concentrations on callus induction rate

[0057] The difference between this example and the previous one is that: after determining the optimal 2,4-D concentration in the previous example, the concentration of 6-BA is screened.

[0058] 6-BA concentration (mg / L) 2,4-D concentration (mg / L) Callus induction rate 0.06 3.5 88.5％～90.3％ 0.05 3.5 94.2％～95.5％ 0.04 3.5 94.2％～96.5％ 0.03 3.5 93.9％～94.6％

[0059] Statistics show that when the concentration ratio of 3.5 mg / L 2,4-D and 0.04 mg / L 6-BA is adopted, the callus induction rate of Elymus sibiricus is the highest, with the contamination rate being 3.4% - 5.2% and the callus induction rate being 94.2% - 96.5%.

[0060] Comparative Example

[0061] The existing patent (authorized announcement number: CN 104304009 B) was used for comparative analysis with the present invention. Among them, the method for inducing Elymus sibiricus callus in the existing patent (authorized announcement number: CN 104304009 B) includes the following steps: Select plump seeds, place them in 3% - 5% sodium hypochlorite for disinfection for 20 min, and wash them 10 times with sterile deionized water; Remove the lemma outside the seeds; Immediately disinfect the seeds after removing the lemma again with 3% - 5% sodium hypochlorite for 20 min, and wash them 10 times with sterile water; After disinfection treatment, cut the embryo of the seeds and place them in a callus induction medium for dark culture. Among them, the MS basal medium is used in callus induction, and 3 mg / L -1 of 2,4-D and 0.1 mg / L -1 of 6-BA are added to the MS basal medium. The situation of the callus on the 7th day of induction is as shown in Figure 1 Figure A, and the situation of the callus on the 14th day of induction is as shown in Figure 2 Figure A.

[0062] After comparative analysis of Figures A and B in Figure 1 and Figures A and B in Figure 2 , it can be clearly observed that the removal of seed-borne fungi in the present invention enables more nutrients in the seeds to be used for the growth of callus, the growth rate of callus becomes faster, and the number of adventitious buds decreases.

[0063] The above are only the preferred embodiments of the present invention, and are not intended to limit the present invention. Any modifications, equivalent replacements, and improvements made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. A method for establishing a callus induction and regeneration system from mature embryos of Elymus sibiricus seeds, characterized in that, The method comprises the following steps: (1) After the old sibiricum seeds collected in the wild were screened, the fungi in the seeds were removed and disinfected; (2) cutting the sterilized seeds longitudinally, and sequentially placing them in an induction medium for induction culture, a subculture medium for subculture culture, a differentiation and proliferation medium for differentiation and proliferation culture, and a rooting medium for rooting culture to obtain seedlings to be differentiated with a root length of 4 to 5 cm; (3) The seedlings to be differentiated are removed from the rooting medium, and the bottle is closed for hardening. The roots are then washed and injected with tap water. The bottle is opened for hardening and sterilized vermiculite is used for culture to obtain complete regenerated plants.

2. The method according to claim 1, wherein In step (1), the treatment for removing seed-borne fungi is: placing the seeds in a drying oven at 65° C. for 14 days.

3. The method according to claim 1, characterized in that In step (1), the disinfection treatment is as follows: the seeds are sequentially soaked in 50% H2SO4 for 10 min, 75% C2H5OH for 2 min, and 50% 84 for 20 min.

4. The method according to claim 1, wherein In step (2), the induction culture process is: cutting the seeds in half longitudinally and placing them in an induction culture medium with a pH of 5.8 to 5.9, and culturing them in the dark at a constant temperature of 26±1°C for 31 to 35 days, wherein the induction culture medium is: a basic culture medium of 3.5 mg / L 2,4-D + 0.04 mg / L 6-BA.

5. The method according to claim 1, wherein In step (2), the subculture process is: placing the seeds in a subculture medium with a pH of 5.8 to 5.9, and culturing for 14 to 16 days at a constant temperature of 26±1°C and in darkness; wherein the subculture medium is: basal medium + 3.5 mg / L 2,4-D + 0.04 mg / L 6-BA.

6. The method according to claim 1, wherein In step (2), the process of differentiation and proliferation culture is as follows: place the seeds in a differentiation and proliferation medium with a pH of 5.7, and culture them at a constant temperature of 26 ± 1 °C under light conditions until the seedling length reaches 2 - 3 cm; wherein, the differentiation and proliferation medium is: basal medium + 2 mg / L 6 - BA + 0.5 mg / L NAA; the light conditions are: light culture, light intensity 4300 - 4800 Lux, and light duration 14 h·d -1 .

7. The method according to claim 1, characterized in that, In step (2), the process of rooting culture is as follows: placing the seedlings in a rooting medium with a pH of 5.7 and culturing them at a constant temperature of 26 ± 1 °C under light conditions until the root length reaches 4 - 5 cm; wherein, the rooting medium is a basal medium, and the light conditions are: intensity 4300 - 4800 Lux, light duration 14 h·d -1 .

8. The method according to any one of claims 4 to 7, characterized in that The basic culture medium is: 4.33 g / L MSsalts powder+30 g / L maltose+3.5 g / L plant gel.

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